Cholesterol is not uniformly distributed in the cell and in membranes. No single mechanism to date explains intracellular cholesterol trafficking. Sterol carrier protein-2 (SCP-2) mediated transfer is the least understood. SCP-2 exists as a family of proteins [primarily 13 (SCP-2), 15 (pro-SCP-2), 29 (SCP-y), and 58 (SCP-x) kDa forms] arising from a single gene and sharing a common 13 kDa C-terminus. They differ in N-terminal targeting sequences, intracellular localization, and presumably function. However, there is as yet no general agreement on whether any SCP-2s even bind cholesterol or on the mechanism(s) whereby these ubiquitous proteins mediate sterol transfer in vitro. With a single exception there is no evidence that any of the SCP-2s function in cholesterol trafficking in intact cells. In this application recombinant SCP-2s will be used to address these issues: 1) Characterize the ligand binding site(s) of recombinant SCP-2s. Although no endogenous ligand copurified with 13 kDa SCP-2, fluorescent and radiolabeled sterols show the presence of a single sterol binding site. These studies will be extended to explore the ligand binding site(s) of the 15, 29, and 58 kDa SCP-2s. 2) Investigate the role of the SCP-2 forms in targeting cholesterol transfer between intracellular membrane fractions and organelles in vitro. The 13 kDa SCP-2 differentially transfers sterol: plasma membranes > microsomes >> mitochondria. Targeting by all SCP-2 forms to specific membranes will be determined. 3) Examine the intracellular distribution and role of the SCP-2 forms on intracellular sterol trafficking and efflux in intact cells. Transfected L-cells expressing 13 or 15 kDa SCP-2s enhanced uptake and esterification of medium derived cholesterol and oxidation of plasma membrane derived cholesterol. The role of all four SCP-2s in cholesterol efflux and intracellular trafficking will be examined in transfected cells. 4) Explore in vitro the mechanism whereby the SCP-2 forms regulate intramembrane cholesterol domain structure. The 13 kDa SCP-2 redistributes cholesterol within model membranes and plasma membranes in vitro to enhance sterol desorption. The effect of all four SCP-2s on membrane sterol domains will be determined in transfected cells. These experiments should yield novel insights into the function of the SCP-2s and provide new data on regulation of cholesterol domain structure. This will contribute to our understanding of diseases involving abnormal cholesterol absorption as well as cholesterol trafficking and/or accumulation (peroxisomal deficiency, cancer, atherosclerosis, aging).